RNeasy FFPE Kit

For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections

Novel method to overcome formalin crosslinking

Efficient release of RNA without compromising integrity

Streamlined protocol providing RNA in just 85 minutes

The RNeasy FFPE Kit is specially designed for purifying total RNA from formalin-fixed, paraffin-embedded tissue sections. Special lysis and incubation conditions reverse formaldehye modification of RNA. In addition, the lysis buffer efficiently releases RNA from tissue sections while avoiding further RNA degradation. The kit also uses DNase and DNase Booster Buffer for optimized removal of genomic DNA contamination. RNeasy MinElute spin columns enable purification of total RNA with elution volumes of as low as 10 μl. Purification can be fully automated on the QIAcube.

The RNeasy FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Reliable results in real-time RT-PCR analysis.

Total RNA was purified from a 10 µm rat lung FFPE sample using the RNeasy FFPE Kit or alternative kits from Supplier EIII, Supplier N, Supplier AIV, and Supplier M. Real-time RT-PCR assays for PGK1 (phosphoglycerate kinase 1) were performed on the Rotor-Gene Q cycler with (+RT) or without (-RT) the reverse transcription step. The high CT value in the -RT sample from the RNeasy FFPE Kit indicates that purified RNA is virtually free of genomic DNA. Therefore the CT value in the +RT sample from the RNeasy FFPE Kit is a reliable measure of RNA expression.

Greater yields of RNA.

RNA was purified from 10 µm FFPE sections of the indicated rat tissues using either the RNeasy FFPE Kit or alternative kits from Supplier N, Supplier AIV, and Supplier M. RNA yields were determined by measuring absorbance at 260 nm.

RNeasy FFPE procedure.

After paraffin is removed from samples, the optimized lysis buffer allows sample lysis with proteinase K digestion in only 15 minutes without sacrificing RNA yields. After lysis, samples are incubated at 80ºC for 15 minutes to reverse formalin crosslinking. Genomic DNA, including small DNA fragments found in FFPE samples, is then effectively removed using DNase and DNase Booster Buffer. Finally, concentrated RNA is purified using RNeasy MinElute spin columns. Since RNA is eluted in a volume of just 14–30 µl, smaller reaction volumes are possible in downstream applications.

Successful real-time RT-PCR analysis.

Total RNA was purified from rat kidney using the RNeasy FFPE Kit. Real-time RT-PCR assays for PGK1 (phosphoglycerate kinase 1) were performed on the Rotor-Gene Q cycler with (+RT) or without (-RT) reverse transcriptase. The -RT curves demonstrated that RNA purified using the RNeasy FFPE Kit was virtually free of genomic DNA.

Recovery of all usable RNA.

RNA purified from 6-month-old FFPE rat liver using the RNeasy FFPE Kit or alternative kits from Supplier AIV, Supplier N, or Supplier M, was analyzed on the Agilent 2100 Bioanalyzer. The peaks indicate that more intact RNA was purified using the RNeasy FFPE Kit than when using alternative kits.

Fixing tissues with formalin leads to RNA–RNA and RNA–protein crosslinking which impairs RNA performance in enzymatic assays. Fixation and embedding conditions can also result in heavily fragmented nucleic acids in FFPE samples. The RNeasy FFPE Kit uses special lysis and incubation conditions to reverse formaldehyde modification of RNA. The lysis buffer efficiently releases RNA from FFPE tissue samples, preventing further RNA degradation. These optimized conditions allow purification of all usable RNA, leading to greater yields from FFPE samples with the RNeasy FFPE Kit than with other methods.

Procedure

The entire RNeasy FFPE procedure can be completed in as little as 85 minutes (see flowchart "RNeasy FFPE procedure"). Sample lysis with proteinase K digestion requires only 15 minutes. After lysis, samples are incubated at 80ºC for 15 minutes. DNase treatment then effectively removes genomic DNA, including small DNA fragments. Finally, concentrated RNA is purified using RNeasy MinElute spin columns, and eluted in a volume of 14–30 µl. Purification can be fully automated on the QIAcube.

Applications

The RNeasy FFPE Kit isolates all RNA molecules longer than 70 nucleotides from FFPE samples, providing usable RNA fragments for numerous downstream applications, including RT-PCR. However, RNA purified from FFPE samples is heavily fragmented and should not be used in downstream applications that require full-length RNA. Some applications may require modifications to allow the use of fragmented RNA (e.g., designing small amplicons for RT-PCR). For cDNA synthesis, either random or gene-specific primers should be used instead of oligo-dT primers.